Among transmissible spongiform encephalopathies (TSEs), the L-type
bovine spongiform encephalopathy (L-BSE) in cattle requires particular
attention for public health. L-BSE is transmitted more efficiently than
is classical BSE among primates (7-3) as well as among transgenic mice
that express human prion protein (PrP) (4,5). We recently reported that
L-BSE was readily transmissible by experimental oral inoculation in a
nonhuman primate species, the grey mouse lemur (Microcebus murinus) (3).
These findings raise the possibility that some human Creutzfeldt-Jakob
disease (CJD) cases might result from exposure to the L-BSE agent;
previous studies highlighted similarities between L-BSE and some human
subtypes (type 2) of sporadic CJD (sCJD) (7,6).

To examine the possible relationship between L-BSE and sCJD, we
evaluated a strain-typing strategy that relies on comparative
transmission characteristics in the Syrian golden hamster and in a
transgenic mouse line (TgOvPrP4) expressing ovine PrP (ARQ allele). Both
of these species are susceptible to L-BSE prions from cattle (7,8). The
transmission of L-BSE, including after a first passage in Microcebus
murinus lemurs (3), was compared with that for the MM2-cortical subtype
of sCJD (9); this subtype was chosen on the basis of a study that
indicated higher levels of molecular similarities of L-BSE with this
sCJD subtype than with the MV2 subtype (7).

The Study

The TSE brain inocula used in this study, conducted during November
2010-December 2011, were derived from 2 natural L-BSE isolates from
France (02-2528 and 08-0074); a lemur injected intracerebrally (i.c.)
with the 02-2528 L-BSE cattle isolate (3); and a human patient with
MM2-cortical sCJD. Consent was obtained for using tissues from the human
patient in research, including genetic analyses. Animal experiments were
performed in the biohazard prevention area (A3) of the Anses-Lyon animal
facilities, in accordance with the guidelines of the French Ethical
Committee (decree 87-848) and European Community Directive 86/609/EEC.

Six-week-old TgOvPrP4 mice and 4-week-old Syrian golden hamsters
were injected i.c. with 20 and 30 [micro]L, respectively, of 10%
(wt/vol) brain homogenates in 5% sterile glucose. Serial passages were
performed in TgOvPrP4 mice by i.c. inoculation of 1% (wt/vol)
homogenates from mice positive for protease-resistant PrP
([PrP.sup.res]). At the terminal stage of the disease, animals were
euthanized, and their brains and spleens were collected for
[PrP.sup.res] analyses by Western blot and for histopathologic studies
(8).

In hamsters, transmission of the MM2-cortical sCJD agent was
inefficient. Clinical signs were absent up to 876 days postinoculation
(dpi) (Table), and disease-associated PrP ([PrP.sup.d]) in brain samples
was not detected by paraffin-embedded tissue blot (PET-blot) (Figure 1,
panel A), immunohistochemical (Figure 1, panel C), or Western blot
(Figure 1, panels E, F) analyses. [PrP.sup.res] was also undetectable in
spleen tissues by Western blot (Table).

In contrast, the L-BSE agent passaged in a lemur was efficiently
transmitted to hamsters, with a mean survival period of 529 [+ or -] 117
dpi, similar to that for L-BSE from cattle (622 [+ or -] 64 dpi)
(Table). PET-blot analysis (Figure 1, panel B) showed widespread
[PrP.sup.res] distribution in the brain; immunohistochemical analysis
(Figure 1, panel D) showed a granular type of [PrP.sup.d] deposition
that redefined the periphery of most of the blood vessels. Western blot
analysis (Figure 1, panels E, F) showed [PrP.sup.res] in the brains of
hamsters inoculated with L-BSE from cattle and lemur and in 1/4 spleens
of hamsters injected with L-BSE passaged in lemur (Table). Brain
[PrP.sup.res] was characterized by low apparent molecular mass
([approximately equals] 19 kDa for the unglycosylated band) associated
with a lack of reactivity toward the N terminal 12B2 antibody, in
contrast to that for the control animal with scrapie (Figure 1, panels
E, F).

In TgOvPrP4 mice, all TSEs were efficiently transmitted, as
confirmed by [PrP.sup.d] accumulation in the mouse brains (Table). After
serial passages in additional TgOvPrP4 mice, the survival periods in
each experiment became considerably shorter (Table; online Technical
Appendix Figure 1, wwwnc.cdc.gov/EID/pdfs/12-0342Techapp.pdf). No
statistically significant differences in results were identified between
the L-BSE sources (p>0.6). Mean survival period decreased to 111 [+
or -] 25 dpi at second passage in mice inoculated with the agent of
MM2-cortical subtype sCJD, which differed significantly from that of
mice inoculated with L-BSE (p<0.0001). A third passage of both cattle
L-BSE and human sCJD did not reduce the survival periods in TgOvPrP4
mice (data not shown).

Western blot analyses of [PrP.sup.res] from mouse brains showed
partially similar features for MM2-cortical sCJD and L-BSE, including
low molecular mass ([approximately equals] 19 kDa for the unglycosylated
band) (Figure 2, panel A) and similar conformational stability of
[PrP.sup.d] after treatment with guanidinium hydrochloride (online
Technical Appendix Figure 2). However, the proportions of
diglycosylated, monoglycosylated, and unglycosylated bands of brain
[PrP.sup.res] differed between sCJD and L-BSE (Figure 2, panel C);
higher proportions of diglycosylated [PrP.sup.res] were found in
sCJD-infected mice (mean 67% of the total signal) compared with
L-BSE-infected mice ([approximately equals] 18% lower; p<0.0001).
[PrP.sup.res] was readily identified in the spleens of TgOvPrP4 mice at
the second passage for sCJD and L-BSE from cattle and at the first
passage for L-BSE from lemur (Table). No significant differences in the
proportions of [PrP.sup.res] glycoforms for sCJD-infected versus
L-BSE-infected mice were observed in the spleens (Figure 2, panel D),
but [PrP.sup.res] was [approximately equals] 0.5 kD higher in mice
injected with sCJD (Figure 2, panel B, arrows).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Histopathologic analysis showed severe vacuolar lesions in TgOvPrP4
mice infected at second passage with sCJD and lemur-passaged L-BSE
(online Technical Appendix Figure 3). However, in sCJD-infected mice,
vacuolar lesions were mostly observed in the anterior parts of the brain
(except the parietal cortex), whereas in mice infected with
lemur-passaged L-BSE, the lesions were more widely distributed,
involving the colliculi and the hypothalamus. In mice infected with sCJD
and lemurpassaged L-BSE, PET-blot analyses showed that most of the
[PrP.sup.res] occurred in the frontal parts of the brain, but the
intensity and appearance of [PrP.sup.res] in the cortex, thalamus, and
hippocampus were distinctly different. Immunohistochemical analyses of
the hippocampus showed [PrP.sup.d] deposition in the dentate gyrus in
sCJD-infected mice, in contrast to a lack of deposition in lemurpassaged
L-BSE-infected mice.

Conclusions

We report the isolation of 2 prion strains derived from L-BSE and
MM2-cortical sCJD after transmission in Syrian hamsters and ovine
PrP-transgenic mice. In hamsters, we did not transmit any disease with
sCJD, but the transmission of L-BSE from lemur was efficient, as
previously reported for L-BSE from cattle (7,77). This result suggests
that L-BSE did not undergo major modifications after this cross-species
transmission and could indicate a clear biologic difference between
MM2-cortical sCJD and L-BSE. We also demonstrated the efficient
transmission of both L-BSE and MM2-cortical sCJD in TgOvPrP4 mice, which
enabled us to compare these diseases in a single model. Unexpectedly,
during serial passages, we observed that the agent of MM2-cortical sCJD
causes a much more rapidly fatal disease. Despite similar molecular
features in sCJD and L-BSE, including the [PrP.sup.res] electrophoretic
mobility and the conformational stability of [PrP.sup.d], sCJD and L-BSE
differed in [PrP.sup.res] glycosylation for the mouse brains and gel
migrations for the mouse spleens. Mice infected with MM2-cortical sCJD
versus those infected with L-BSE also showed distinct lesion profiles
and [PrP.sup.d] distribution, which confirms clear biologic differences
between these diseases.

Although only 1 case of sCJD of a unique molecular subtype was
examined in our study, our observations do not support the hypothesis of
a causal relationship between L-BSE and this human sCJD subtype. Our
study thus encourages further investigations using the proposed bioassay
approach for a more complete evaluation of possible relationships
between L-BSE and human prion diseases.

Acknowledgments

We thank the staff of the Plateforme d'Experimentation Animale
of Anses-Lyon for excellent animal care; Mikael Leboidre and Jean-Michel
Bridon for histotechnical assistance; Dominique Canal and Claire
Aufauvre for biochemical assistance; and Francoise Didier and Nathalie
Streichenberger for characterization of the human sporadic CJD case.

S.N. was supported by grants from the Agence Nationale de Securite
Sanitaire and the Fondation pour la Recherche Medicale.

Mr Nicot is a PhD student at the Agence Nationale de Securite
Sanitaire in Lyon. His primary research interests include
characterization of the infectious agents and prion protein during
intra- and interspecies transmission of animal and human prion diseases.